EP2360238A1 - Souche de sphingomonas déficiente pour la génération de pigments jaunes et son application pour la fabrication de gomme gellane - Google Patents

Souche de sphingomonas déficiente pour la génération de pigments jaunes et son application pour la fabrication de gomme gellane Download PDF

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Publication number
EP2360238A1
EP2360238A1 EP10818224A EP10818224A EP2360238A1 EP 2360238 A1 EP2360238 A1 EP 2360238A1 EP 10818224 A EP10818224 A EP 10818224A EP 10818224 A EP10818224 A EP 10818224A EP 2360238 A1 EP2360238 A1 EP 2360238A1
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EP
European Patent Office
Prior art keywords
gellan gum
strain
acyl gellan
fiber
sphingomonas strain
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP10818224A
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German (de)
English (en)
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EP2360238A4 (fr
EP2360238B1 (fr
Inventor
Xuechang Wu
Rongming Wu
Ou LI
Liang Zhu
Yamin Chen
Chaodong Qian
Mei Chen
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Zhejiang University ZJU
Zhejiang DSM Zhongken Biotechnology Co Ltd
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Zhejiang University ZJU
Zhejiang DSM Zhongken Biotechnology Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Definitions

  • the present invention is related to a strain, i.e., the Sphingomonas strain ZD001 ( Sphingomonas sp. ZD001) which is characterized as being yellow pigments generation deficient and being capable of producing gellan gum having the normal quality, and to the application of this strain in the preparation of gellan gum via the fermentation of microorganism.
  • Sphingomonas strain ZD001 Sphingomonas sp. ZD001
  • Gellan Gum is a microbial extracellular polysaccharide, produced by a Sphingomonas strain (S phingomonas paucimobilis ) via aerobic fermentation. This product was successfully developed by CP Kelco U.S., Inc. in 1978. It is another microbial extracellular polysaccharide after xanthan, which is non-toxic and safe, and shows good physicochemical properties. It was approved to be used in food products in Japan, as early as in 1988. The FDA of the USA government approved its use in foodstuff in 1992. In our country, it was approved to be used as a thickener or a stabilizer for food products in 1996. And there are more than ten of other countries, which have approved its use as an additive for food products.
  • gellan gum is increasingly and broadly used in food industry, medicine industry, chemical industry and other industrial fields, as a new emulsifier, a suspending agent, a thickener, a stabilizer, a gelling agent, a slow-released agent, a film-forming material and etc., due to its unique good properties. This shows a huge prospect of its applications being broadened.
  • Gellan gum is comprised of ⁇ -1,3-D-glucose, ⁇ -1,4-D-glucuronic acid and ⁇ -1,4-L-rhamnose in the mole ratio of 2:1:1, with the linking order of a ⁇ -1,3-D-glucose, a ⁇ -1,4-D -glucuronic acid, a ⁇ -1,3-D-glucose, and an ⁇ -1,4-L-rhamnose to form the tetrasaccharide unit.
  • acyl groups linked to the framework of long chains of sugars formed by the polymerization of tetrasaccharide units.
  • the molecular weight of gellan gum normally is about 5 ⁇ 10 5 -1 ⁇ 10 6 daltons.
  • Gellan gum mainly exists in two forms, i.e., the non-deacylated high acyl gellan gum (also called as native gellan gum), and the physically and/or chemically artificially-deacylated low acyl gellan gum.
  • the high acyl or native gellan gum there are two kinds of acyl groups, namely, acetyl and glyceroyl.
  • the acetyl groups are linked to the C 6 position of the first glucose residue while the glyceroyl groups are linked to the C 2 position of the same glucose residue.
  • the low acyl gellan gum is a product substantially free of acyl groups, produced from high acyl gellan gum by a deacylation treatment. So, the molecules of gellan gum have various molecule weights showing large differences, depending upon whether the molecules are deacylated or not and how much degree the molecules are deacylated. At present, the low acyl gellan gum with a relatively low molecular weight has the most applications in food industry.
  • the gellan gum-producing bacterial strains currently used in production can co-produce the by-products through metabolism during fermentation process, i.e., the yellow pigments (mainly the pigments carotinoids), which not only competes the carbon source with gellan gum, but only makes the fermentation broth yellowing.
  • the yellow pigments mainly the pigments carotinoids
  • the preparation of gellan gum particularly in the preparation of high acyl gellan gum, in order to remove the yellow pigments from the colloid, it needs to increase the amount of ethanol or isopropanol used in decoloration by extraction (in general, ethanol or isopropanol is used at the amount based on volume of 3 times of the volume of the fermentation broth), and the time for the operation. This reduces the efficiency of extraction and purification, leading to an increase on costs.
  • One aim of the present invention is to provide a yellow-pigments generation-deficient strain, but being capable of producing gellan gum with normal quality, i.e., a sphingomonas strain, called as ZD001 ( Sphingomonas sp. ZD001).
  • ZD001 Sphingomonas sp. ZD001
  • the ZD001 strain has been deposited at China Center for Type Culture Collection (CCTCC), the address of the center: Wuhan University, Wuhan, China, Post Code 430072; and the preservation date of the strain: September 10, 2009; the preservation serial number: CCTCC No: M209198.
  • CCTCC No: M209198 the strain is called as ZD001 (CCTCC NO: M 209198) strain.
  • the said ZD001 (CCTCC NO: M 209198) strain has a 16S rDNA with the following sequence:
  • the said ZD001 (CCTCC NO: M 209198) strain is characterized in that: Gram-negative bacillus; non-spore-forming ; straight rod in shape; colonies on nutrient agar plates showing the ivory-white color; positive in oxidase test; positive in catalase test; obligate aerobe; decomposing glucose, fructose, xylose and saccharose; positive in starch hydrolysis test; incapable of liquidizing gelatin; no growth at 43°C; the size of cells being in the range of 1.5 ⁇ 5.0 ⁇ m ⁇ 0.8-1.0 ⁇ m; and no production of yellow pigments.
  • the present invention also relates to the use of the said ZD001 (CCTCC NO: M 209198) stain in the preparation of gellan gum via microorganism fermentation.
  • the said ZD001 (CCTCC NO: M 209198) strain is activated by routine methods, the seed obtained thereof is cultured and the cultured seed is inoculated into the fermentation medium usually suitable for the growth of Sphingomonas strains. Then, at the temperatures ranging 28 ⁇ 32°C and pH 6.8 ⁇ 7.2, the culture is cultured on a shaker for 32-60 hrs and thereby the high acyl gellan gum-containing fermentation broth is obtained.
  • the fermentation broth can be directly separated and extracted to obtain the high acyl gellan gum product, or it can be subjected to a deacylation treatment to prepare the low acyl gellan gum.
  • the method is conducted as follows.
  • the pH value of the said high acyl gellan gum-containing fermentation broth is adjusted to 5.0 ⁇ 6.0, and the temperature is raised up to 50°C ⁇ 70°C (preferably, 60°C), and the temperature is kept constant for 30 min-2 h (preferably, 1 h).
  • the temperature is lowered to 40°C ⁇ 50°C, the pH is adjusted to 7.0, and then, lysozyme and alkaline proteinase are added and the temperature is kept as it was for 1 h - 3 h (preferably, 2 h) to remove the proteins.
  • a flocculating agent solution is added to conduct the flocculation (the amount of the flocculating agent solution is about 5% of the fermentation broth in volume after the proteins have been removed).
  • the solid material is dried (at 90°C) to obtain the said high acyl gellan gum.
  • the flocculating agent is one or a mixture of more than one, of the following substances (the concentration usually is 20%, w/v): CaCl 2 , MgCl 2 , NaCl, and KCl.
  • the method is conducted as follows. Into the said high acyl gellan gum-containing fermentation broth, a solution of an alkali metal chloride or an alkali earth metal chloride (usually having a concentration of 20%, w/v) is added and the pH is adjusted to 11.0, then a solid-liquid separation (a solid-liquid separation via a plate-and-frame device) is conducted so that a fiber-like material 1 is obtained.
  • an alkali metal chloride or an alkali earth metal chloride usually having a concentration of 20%, w/v
  • a solid-liquid separation a solid-liquid separation via a plate-and-frame device
  • the so-obtained fiber-like material 1 is mixed with water in the ratio of 1 : 4 ⁇ 6 in volume, the pH is adjusted to 2.5 ⁇ 4.0 (preferably, pH 3.0), a washing is conducted for 15 min ⁇ 1 h (preferably, 30 min), and a filter press is given so that a fiber-liked material 2 is obtained.
  • the so-obtained fiber-like material 2 is mixed evenly with water in the ratio of 1 : 9 ⁇ 12 in volume, and the temperature is raised up to 80°C ⁇ 90°C (preferably, 83°C ⁇ 87°C), and a basic agent is added to adjust the pH to 9.5 ⁇ 10.5 (preferably, pH 9.8-10.2).
  • the reaction is conducted for 8 min ⁇ 15 min (preferably, 10 min ⁇ 12 min).
  • the pH of the reaction liquid is adjusted to the neutral condition and a filtration aid is added (the amount of addition is 1 ⁇ 3%, w/v, preferably 2%) and a filtration is conducted.
  • a flocculating agent is added (preferably, 10% in mass concentration, and the volume added is 5% of the filtrate volume) to conduct a flocculation.
  • a separation is given and the solid material obtained is dried to prepare the said low acyl gellan gum.
  • the said alkali metal chloride or alkali earth metal chloride is one selected from the following list or a mixture of more than two substances selected from the following list: CaCl 2 , MgCl 2 , NaCl, and KCl (preferably, CaCl 2 ).
  • the said basic agent is one selected from the list or a mixture of more than two substances selected from the list: NaOH, KOH, and Na 3 PO 4 .
  • the filtration aid is diatomite, perlite or a mixture thereof.
  • the flocculating agent is one selected from the list or a mixture of two or more selected from the list: CaCl 2 , MgCl 2 , NaCl, and KCl (preferably, KCl).
  • the main beneficial effects of the present invention are that: a yellow pigments generation deficient Sphingomonas strain ZD001 (CCTCC NO: M 209198) is provided, whose fermentation broth does not contain yellow pigment(s) and has the color of milky white; during the extraction, the amount of ethanol or isopropanol for use is reduced; and the processing steps are simplified. All these beneficial effects improve the yield, and lower the production costs. It is helpful in the industrial production of high-quality gellan gum.
  • composition of YM agar medium are 0.30% of yeast extract, 0.30% of malt extract, 0.50% of peptone, 1.00% of glucose, 1.50% of agar, and the solvent distilled water.
  • the concentrations in the medium refer to the mass-to-volume percent concentrations.
  • the concentration 1% means that there is 1 g of the substance existing in the 100 ml medium.
  • a yellow pigments generation deficient strain i.e., ZD001 (CCTCC NO: M 209198) strain.
  • This strain is a Gram-negative bacillus; does not produce spores; and has the cells in the shape of straight rod.
  • the colony on nutrient agar medium plate has the color of ivory white.
  • the strain is positive in oxidase test, positive in catalase test, and is an obligate aerobe.
  • the stain can decompose glucose, fructose, xylose and saccharose.
  • the strain is positive in starch hydrolysis test, and does not liquidize the gelatin.
  • the stain cannot grow at 43°C.
  • the dimension of the bacterial bodies is 1.5-5.0 ⁇ m ⁇ 0.8-1.0 ⁇ m. The stain does not produce yellow pigment(s).
  • YPG slant medium glucose 2.00%, peptone 0.50%, yeast extract 0.30%, agar 1.50%, and the solvent being distilled water, pH 7.2;
  • the final concentrations for the first class culture medium are: yeast extract 0.20%; beef extract 0.30%; peptone 0.50%; potassium chloride 0.10%, and the solvent being distilled water, pH 7.2;
  • the second class seed medium glucose 1.50%; yeast extract 0.50%; peptone 0.50%; potassium dihydrogen phosphate 0.06%; dipotassium hydrogen phosphate 0.06%; magnesium sulfate 0.06%, and the solvent being distilled water, pH 7.2;
  • the fermentation medium glucose 3.00%; yeast extract 0.05%; peptone 0.30%; potassium dihydrogen phosphate 0.06%; dipotassium hydrogen phosphate 0.10%; magnesium sulfate 0.06%; and the solvent being distilled water, pH 7.2.
  • Example 3 the preparation process for preparing high acyl gellan gum
  • Example 4 The preparation process for preparing deacylated gellan gum

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Biomedical Technology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Polysaccharides And Polysaccharide Derivatives (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
EP10818224.7A 2009-09-25 2010-08-13 Souche de sphingomonas déficiente pour la génération de pigments jaunes et son application dans la fabrication de gomme gellane Active EP2360238B1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN2009101530057A CN101665778B (zh) 2009-09-25 2009-09-25 黄色素生成缺陷鞘脂单胞菌及其在结冷胶生产中的应用
PCT/CN2010/001228 WO2011035530A1 (fr) 2009-09-25 2010-08-13 Souche de sphingomonas déficiente pour la génération de pigments jaunes et son application pour la fabrication de gomme gellane

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EP2360238A1 true EP2360238A1 (fr) 2011-08-24
EP2360238A4 EP2360238A4 (fr) 2013-01-09
EP2360238B1 EP2360238B1 (fr) 2014-01-15

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US (1) US8685698B2 (fr)
EP (1) EP2360238B1 (fr)
JP (1) JP5765859B2 (fr)
CN (1) CN101665778B (fr)
WO (1) WO2011035530A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3483280A1 (fr) * 2017-11-10 2019-05-15 Zhejiang Dsm Zhongken Biotechnology Co., Ltd Procédé de fermentation pour la production de gomme gellane

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CN101665778B (zh) * 2009-09-25 2012-03-28 浙江大学 黄色素生成缺陷鞘脂单胞菌及其在结冷胶生产中的应用
CN102311508B (zh) * 2010-07-09 2014-05-14 郸城财鑫糖业有限责任公司 一种透明型高酰基结冷胶的提取工艺
CN102747016A (zh) * 2012-06-28 2012-10-24 福建省农业科学院农业生物资源研究所 一种结冷胶高效生产菌株及其应用
SG11201505388QA (en) * 2013-01-31 2015-08-28 Glaxo Group Ltd Method of producing a protein
CN103509845B (zh) * 2013-08-09 2015-07-08 新疆阜丰生物科技有限公司 一种高酰基结冷胶的提取方法
CN103421718B (zh) * 2013-08-09 2015-11-11 浙江大学 一种少动鞘氨醇单胞菌菌株及其应用
CN104193841B (zh) * 2014-08-07 2016-08-24 新疆阜丰生物科技有限公司 一种低成本低酰基透明型结冷胶提取工艺
CN108690143A (zh) 2017-04-07 2018-10-23 帝斯曼知识产权资产管理有限公司 一种高透明低酰基结冷胶的生产方法
CN113151050B (zh) * 2021-03-10 2022-10-11 南京工业大学 一株鞘氨醇单胞菌及其应用
CN113698988B (zh) * 2021-07-27 2024-04-26 长寿花食品股份有限公司 一种营养玉米油的生产工艺
CN113956372B (zh) * 2021-11-05 2022-07-26 南开大学 一种高酰基三赞胶及其生产菌株的分子标记和应用
CN113881423B (zh) * 2021-11-10 2022-10-04 南京工业大学 一种温敏型杂多糖聚合物在提高石油采收率中的应用
CN114214234B (zh) * 2021-12-21 2023-09-26 江苏大学 一种低分子量结冷胶生产菌株及其筛选方法与应用

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3483280A1 (fr) * 2017-11-10 2019-05-15 Zhejiang Dsm Zhongken Biotechnology Co., Ltd Procédé de fermentation pour la production de gomme gellane
US11149293B2 (en) 2017-11-10 2021-10-19 Zhejiang Dsm Zhongken Biotechnology Co. Ltd Fermentation method for producing gellan gum

Also Published As

Publication number Publication date
US8685698B2 (en) 2014-04-01
EP2360238A4 (fr) 2013-01-09
EP2360238B1 (fr) 2014-01-15
JP2013505709A (ja) 2013-02-21
JP5765859B2 (ja) 2015-08-19
US20110281308A1 (en) 2011-11-17
WO2011035530A1 (fr) 2011-03-31
CN101665778A (zh) 2010-03-10
CN101665778B (zh) 2012-03-28

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